Superparamagnetic monodisperse particles

ABSTRACT

PCT No. PCT/FR97/00912 Sec. 371 Date Jan. 15, 1998 Sec. 102(e) Date Jan. 15, 1998 PCT Filed May 23, 1997 PCT Pub. No. WO97/45202 PCT Pub. Date Dec. 4, 1997The superparamagnetic monodispersed particles comprise a core of a first polymer, an internal layer of a second polymer coating the core and in which a magnetic material is distributed, and an external layer of a third polymer coating the magnetic layer and capable of interacting with at least one biological molecule. At least the second polymer is heat sensitive and has a predetermined lower critical solubility temperature (LCST) of 15-65 DEG  C. These particles may be used to isolate at least one biological molecule from a liquid specimen.

The present invention relates to monodisperse superparamagneticparticles, to their process of production and to their uses, inparticular in biology for the isolation of biological molecules.

Monodisperse superparamagnetic particles are disclosed in the prior art.By way of illustration, documents EP-0,106,873 and EP-0,446,260 describemonodisperse superparamagnetic particles comprising a porous core basedon a polystyrene/divinylbenzene copolymer in which particles of magneticiron oxide are incorporated, and a functionalized external layer capableof interacting with nucleic acid probes.

According to the process for producing the particles described in thesedocuments, the magnetic iron oxides are incorporated by precipitation ofthe corresponding salts. This limits the content of magnetic fillerincorporated and makes it possible to obtain the magnetic filler only asa monolayer.

Document EP-0,585,868 describes magnetic particles consisting of a corebased on a first polymer and on a magnetic layer covering the core,which layer consists of a second polymer, in which the ferrite-basedmagnetic material is distributed, and is capable of interacting with anantigen or an antibody, the magnetic material being deposited byprecipitation of iron salts.

The magnetic material incorporated is directly exposed to the subsequenttreatments of the particles and as a result there is a loss of fillerduring use of the particles. This may lead to problems, in particular toproblems of enzyme inhibition and of denaturation of biologicalentities.

According to the invention, particles which are superparamagnetic andwhich have a magnetic filler distributed very homogeneously are used,the content of which may vary between 1 and 80%, in particular from 25to 80% by weight with respect to the polymer(s) constituting theparticles. The present invention makes it possible to achieve highcontents of incorporated magnetic filler, in particular since theprocess employed makes it possible to distribute the magnetic filler inthe form of multilayers. This results in a considerable advantage,namely the possibility of effective separation of the particles of theinvention from the specimen, without having recourse to the combinedaction of another separation technique, such as flocculation.

The magnetic filler is in the form of nano-particles which areincorporated in the particles in a substantially irreversible manner,i.e. without loss by release, whatever the subsequent treatments whichare applied to them, in particular in the specimen, namely rinsingoperations, variations in temperature, in pH, etc.

The properties of the particles of the invention result from theirparticular structure and particular composition and more specificallyfrom the presence of a constituent heat-sensitive polymer at least forsupporting the magnetic material.

A process for obtaining magnetic particles, comprising a core based on afirst polymer consisting of a polystyrene and in which a magneticmaterial is distributed, and a hydrophilic layer covering the core,based on a heat-sensitive polymer consisting ofpoly(N-isopropylacrylamide), is known from the article by A. KONDO, (A.KONDO, H. KAMURA and K. HIGASHITANI (1994), Appl. Microbiol.Biotechnol., 41, 99-105). The process described comprises the followingsteps:

in a first step, for obtaining the magnetic core, the magnetic materialis brought into contact with styrene in the presence of a polymerizationinitiator and then

in a second step, for obtaining the hydrophilic layer, the core obtainedis brought into contact with N-isopropylacrylamide and methacrylic acid,in the presence of the above polymerization initiator.

Bovine serum albumin is fixed to the particles thus obtained in ordersubsequently to isolate antibodies directed against the bovine albuminserum present in a specimen.

The drawback of these particles occurs in the step for separating them:these particles incorporate a small amount of magnetic filler andfurthermore have sizes which vary greatly, considerably limiting theeffectiveness of an applied magnetic field for separating the particles.Thus, in order to ensure as efficient as possible a separation of theseparticles in the specimen, the authors have employed thermoflocculationin which the temperature of the specimen is increased, this having theeffect of completing the action of a magnetic field.

The requirement of an additional separation technique results from theparticles obtained which have the following drawbacks:

low contents of incorporated magnetic filler,

non-homogeneous distribution of the magnetic filler and

formation of non-monodisperse particles.

The particles of the invention are intended for isolating biologicalmolecules essentially by applying a magnetic field, independently of anyvariations in temperature, pH or ion strength.

The monodisperse superparamagnetic particles of the invention have apredetermined size of between 0.1 and 10 μm and comprise:

a core based on a first polymer,

an internal layer, called the magnetic layer, covering the core, basedon a second polymer, in which layer a magnetic material is distributed,and

an external layer, called the encapsulation layer, optionallyfunctionalized, covering the magnetic layer, based on a third polymerand capable of interacting with at least one biological molecule,

at least the second polymer being heat-sensitive and having apredetermined lower critical solubility temperature (LCST) of between 15and 65° C. and preferably between 25 and 50° C.

Advantageously, the second polymer is obtained by polymerization of (1)a water-soluble monomer of acrylamide or of an acrylamide derivative,such as N-isopropylacrylamide (NIPAM), (2) at least one crosslinkingagent, such as N,N-methylenebisacrylamide and (3) at least onefunctional, cationic and water-soluble monomer different from themonomer (1), such as 2-aminoethyl-methacrylate chloride. A secondpreferred polymer is PNIPAM [poly(N-isopropylacrylamide)].

The first polymer may be identical to the second polymer or differentfrom the second polymer and, in the latter case, the first polymer willpreferably be a polymer having a hydrophobic character and in particulara polystyrene or a polymethyl methacrylate.

The third polymer is a polymer compatible with the second polymer and isselected from hydrophilic polymers, in particular acrylamide derivativesand preferably PNIPAM. When this third polymer is functionalized, itbears one or more functional groups selected from carboxylic, aldehyde,thiol and amine functional groups.

A second subject of the invention is a process for obtaining particlesas defined above, which comprises the following steps:

in a step (a), called the step for obtaining the first polymer, thefirst polymer is obtained by polymerization of the suitable monomer ormonomers,

in a step (b), called the step for obtaining the second polymer, a solof the second polymer is obtained by polymerization in aqueous phase of(1) a water-soluble monomer of acrylamide or of an acrylamidederivative, (2) at least one crosslinking agent and (3) at least onefunctional, cationic and water-soluble monomer different from themonomer (1),

in a step (c), called the step for adsorption of the magnetic material,the magnetic material is brought into contact with the first and secondpolymers at a temperature below the LCST of the second polymer,

in a step (d), called the step for obtaining the magnetic layer, thereaction mixture obtained in (c) is raised to a temperature above theLCST of the second polymer, and

in a step (e), called the encapsulation step, the mixture obtained in(d) is brought into contact, in aqueous phase, with the monomer ormonomers suitable for obtaining the third polymer by polymerization.

Steps (a) and (b) are, according to a variant of the process, carriedout simultaneously, in particular but not restrictively when the firstpolymer is identical to the second polymer.

For step (b) and optionally step (a), the polymerization reactants arepreferably selected as follows:

the monomer (1) is preferably selected from N-alkylacrylamides andN,N-dialkylacrylamides, in particular from N-isopropylacrylamide,N-ethylmethacrylamide, N-n-propylacrylamide, N-n-propylmethacrylamide,N-isopropylmethacrylamide, N-cyclopropylacrylamide,N,N-diethylacrylamide, N-methyl-N-isopropylacrylamide,N-methyl-N-n-propylacrylamide, the monomer (1) preferably beingN-isopropylacrylamide (NIPAM),

the functional monomer or monomers (3) are selected from acrylic andmethacrylic derivatives, 2-aminoethyl methacrylate (AEM) chloride,N-vinylpyridine derivatives, trialkylammonium derivatives andisothiouronium chloride derivatives and optionally,

the crosslinking agent (2) is water-soluble and is selected fromN,N-methylenebisacrylamide (MBA) and ethylene glycol dimethacrylate.

For step (c), the adsorption of the magnetic material on the secondpolymer results from electrostatic interactions between particles ofopposite charges. The reaction medium for the adsorption is an aqueousphase, the ion-strength and pH parameters of which are controlled.

The invention furthermore relates to applications of the particlesdefined hereinabove. Thus, the particles can be used in particular tocapture and then separate, in a liquid specimen, at least one biologicalmolecule, in particular selected from proteins, antibodies, fragments ofantibodies, antigens, polypeptides, enzymes, haptens, nucleic acids andfragments of nucleic acids. The biological molecule or molecules arefixed to the particles, directly or indirectly, by adsorption or bycovalent bonding, and, in the latter case, via a ligand for example.

Examples of particular uses of the particles of the invention are asfollows:

use as a tracer after magnetic concentration on a solid phase:

in this case, the particle is counted after scanning the surface usingan atomic force microscope tip or after direct microscopic observation,or using a camera; the magnetic particles may be detected because oftheir metallic charge and may be measured using a magnet-ometer or anyother system of the type for reading a credit card; in order to make iteasier to concentrate the particles on the surface, a permanent magnetor an electromagnet may be placed below or above the surface used forthe detection; magnetic concentration will preferably take place if alimited number of particles is used, for example the quantity sufficientto cover from one to ten times the surface area if the process is staticand optionally a greater quantity if the concentration is dynamic, bycontrolled circulation of liquid on the reactive surface;

uses of the particles coupled to an appropriate biological ligand withinan agglutination reaction protocol; the size of the particles may bemonitored directly in the medium or after magnetic attraction;

use of the particles for carrying reactants into a device of thecapillary type, a set of electromagnets allowing the particles to bemoved;

use of the particles for creating preferred channels and/or forobstructing liquid distribution channels;

use of the particles for transporting therapeutic substances right totheir targets:

the active principle is adsorbed or transiently coupled by covalency tothe surface of the particle and a suitable magnetic field is applied inorder to move the combination of particle and therapeutic substance.

Another subject of the invention is a process for isolating, in a liquidspecimen, at least one biological molecule, in which:

particles according to the invention are used,

said specimen is brought into contact with said particles, byincubation,

a magnetic field is applied to the mixture obtained and

the particles are separated from the specimen.

Of course, the separation of the particles, which forms the subject ofthis latter process, is different from the separation of the biologicalmolecules which is included in the notion of isolation. In the formercase, it is a question of separating, from the liquid specimen, thoseparticles to which the biological molecule or molecules are fixed, bythe action of a magnetic field.

Lastly, a final subject of the invention is a reactive means forisolating biological molecules comprising a dispersion, in aqueousmedium, of particles as defined above.

Before describing the present invention in greater detail, certain termsemployed in the description will be defined.

The expression superparamagnetic particles is understood to meanparticles containing particles of a magnetic material, guaranteeing,after removal of the magnetic field, the absence of any remanentmagnetization.

The expression monodisperse particles is understood to mean particleshaving approximately the same size, and more specifically a size whichvaries by at most 5% with respect to a given and chosen average size.

The expression "to isolate a biological molecule", according to theinvention, comprises the separation and detection of a biologicalmolecule, the enrichment of a fraction within a biological molecule,using a specific or non-specific method of isolation, qualitativelyand/or quantitatively, directly or indirectly, for example via a ligandfixed to the particles.

EXAMPLE 1 Production of the First and Second Polymers

1) The first and second polymers are different, the first being apolystyrene and the second being PNIPAM

The production processes detailed below are used [lacuna] radicalpolymerization in heterogeneous medium, using the following initialreactants:

for the first polymer:

the monomer is styrene (St) (Janssen),

for the second polymer:

the monomer (1) is N-isopropylacrylamide (NIPAM) (Kodak),

the crosslinking agent is N,N-methylenebisacrylamide (MBA) (Aldrich),

the functional monomer (3) is 2-aminoethyl methacrylate (AEM) chloride(Kodak),

the polymerization initiator is 2,2'-azobis (amidinopropane) chloride(V50) (Wako), and NaCl was used to adjust the ion strength.

1.1) Closed-reactor (Batch) Polymerization

All the aforementioned monomers are introduced into the reactor beforethe start of the polymerization reaction together with the otherreactants and without subsequent addition. This method proves to be veryeffective for copolymerizing a mixture of hydrophobic and hyprophilicmonomers since the hydrophobic monomer (St) mainly forms the core andthe hydrophilic monomer (NIPAM) forms the layer covering the core, ifthe polymerization takes place in aqueous phase.

The synthesis is carried out in a 250 ml reactor with constant stirringat 200 revolutions/minute and in an inert atmosphere of nitrogen. Thewater used, boiled and degassed under nitrogen for two hours, isintroduced into the reactor, which is thermostatted at 70° C., and leftunder a gentle stream of nitrogen for 15 minutes so as to remove anytraces of oxygen. The monomers (St, NIPAM) are introduced and degassedfor a further 15 minutes before adding the initiator V50.

Formulation of the reaction mixture:

    ______________________________________                                               Reactants     Mass                                                     ______________________________________                                               water         200 ml                                                     St 18 g                                                                       NIPAM 2.06 g                                                                  V50 0.2053 g                                                                ______________________________________                                    

Characteristics of the colloidal dispersion obtained:

    ______________________________________                                        (a) diameter at 20° C.                                                                         376 nm                                                  (b) diameter at 50° C. 330 nm                                          (c) diameter using TEM 326 nm                                                 (d) density of the dispersed phase 10 mmol/g                                ______________________________________                                         (a) diameter measured by dynamic light scattering at 20° C.            (b) diameter measured by dynamic light scattering at 50° C.            (c) diameter measured using transmission electron microscopy                  (d) density of the dispersed phase expressed in mmol (amine)/g of polymer

1.2 Polymerization on a Seed

This method consists in introducing the monomer or monomers (1) and/or(3) into a reactor containing the colloidal dispersion 1.1) alreadyformed and perfectly characterized, in the presence of the crosslinkingagent MBA. The monomer or monomers (1) and/or (3) may be added to theseed, in a single step or semi-continuously.

The polymerization reaction is carried out in a 100 ml reactor, at atemperature of 70° C., with stirring at 200 revolutions/minute. Theduration of the polymerization reaction is 19 hours.

Formulation of the reaction mixture identified by the reference(PS131/132):

    ______________________________________                                        Reactants              Mass (g)                                               ______________________________________                                        Polymer according to 1.1                                                                             1.26                                                     NIPAM 0.77                                                                    MBA 0.06                                                                      AEM 0.06                                                                      V50 0.018                                                                   ______________________________________                                    

Characteristics of the sol obtained:

    ______________________________________                                        (a) diameter at 20° C.                                                                         610 nm                                                  (b) diameter at 50° C. 450 nm                                          (c) diameter using TEM 305 nm                                                 (d) density of the dispersed phase 19 mmol/g                                ______________________________________                                         (a) diameter measured by dynamic light scattering at 20° C.            (b) diameter measured by dynamic light scattering at 50° C.            (c) diameter measured using electron microscopy                               (d) density of the dispersed phase expressed in mmol (amine)/g of polymer

2) The First and Second Polymers are Identical and are PNIPAM

The initial reactants are those which were selected in 1) for the secondpolymer.

2.1) Batch Polymerization (or Closed-reactor Process)

The monomer (1) (NIPAM), the functional monomer (3) (AEM) and thecrosslinking agent (MBA) are introduced together in a single step beforethe polymerization is initiated by adding the initiator (V50). Theduration of the polymerization is 30 min.

Formulation of the polymer obtained, identified by the referencePNIPAM42:

    ______________________________________                                        Total volume of boiled and                                                                           250 ml                                                   degassed water                                                                NIPAM 48.51 mmol                                                              MBA 3 mmol                                                                    AEM 0.48 mmol                                                                 V50 0.30 mmol                                                                 Temperature 70° C.                                                   ______________________________________                                    

The characteristics of the polymer obtained after polymerization aregiven in the following table:

    ______________________________________                                        diameter.sup.(a), DLS 20° C. to 20° C.                                                  292 nm                                                  [sic]                                                                         diameter.sup.(b), DLS size at 40° C. 164 nm                            diameter.sup.(c), TEM 129 nm                                                  concentration of AEM.sup.(d) 14.1 μmol/g                                    of polymer                                                                   LCST.sup.(e) 31.5° C.                                                  CCC.sup.(f) at 20° C. 1.00 mol/l                                     ______________________________________                                         .sup.(a) diameter measured by dynamic light scattering at 20° C.       .sup.(b) diameter measured by dynamic light scattering at 40° C.       .sup.(c) diameter measured using electron microscopy                          .sup.(d) density of the dispersed phase expressed in mmol (primary            amine)/g of polymer.                                                          .sup.(e) lower critical solubility temperature (LCST) determined by           measuring the turbidity as a function of temperature                          .sup.(f) critical coagulation concentration (CCC) at 20° C.       

2.2) Semi-continuous Polymerization

The monomer (3) is introduced in two stages, at 3 min and at 6 minrespectively, into the reactor which already contains the monomer (1),the crosslinking agent (2) MBA and the initiator V50, in the course ofpolymerization. This addition may be carried out at a constant rate ofinjection (polymerization by continuous addition) or else withwell-controlled addition at regular intervals (semi-continuouspolymerization). The aim of this polymerization method is to increasethe incorporation of functional monomer(s) (3) without increasing thepercentage of water-soluble polymer in the reaction mixture.

Formulation of the polymer obtained, identified by the referencePNIPAM45:

    ______________________________________                                        Total volume of boiled and                                                                           250 ml                                                   degassed water                                                                NIPAM 48.51 mmol                                                              MBA 3 mmol                                                                    AEM 0.48 mmol                                                                 V50 0.30 mmol                                                                 Temperature 70° C.                                                     Additions between 3                                                            and 6 min.                                                                 ______________________________________                                    

The characteristics of the polymer PNIPAM45 obtained afterpolymerization are given in the following table:

    ______________________________________                                        diameter.sup.(a), DLS 20° C. to 20° C.                                                  823 nm                                                  [sic]                                                                         diameter.sup.(b), DLS size at 40° C. 530 nm                            diameter.sup.(c), TEM 327 nm                                                  concentration of AEM.sup.(d) 10.0 μmol/g                                    of polymer                                                                   LCST.sup.(e) 32° C.                                                    CCC.sup.(f) at 20° C. 1.00 mol/l                                     ______________________________________                                         .sup.(a) diameter measured by dynamic light scattering at 20° C.       .sup.(b) diameter measured by dynamic light scattering at 40° C.       .sup.(c) diameter measured using electron microscopy                          .sup.(d) density of the dispersed phase expressed in mmol (primary            amine)/g of polymer.                                                          .sup.(e) lower critical solubility temperature (LCST) determined by           measuring the turbidity as a function of temperature                          .sup.(f) critical coagulation concentration (CCC) at 20° C.       

2.3) Polymerization on Seed

This method consists in introducing the monomer or monomers (1) and/or(3) into a reaction medium containing a sol of the polymer producedbeforehand according to 2.1 and perfectly characterized.

Formulation of the reaction mixture:

A volume of 40 ml of seed 2.1 with a concentration of 4.5 g per 100 mlis used. The reactants were added, diluted in a volume of 5 ml of water.The molar percentages of NIPAM, MBA and V50 added in the second step areidentical to those of the seed as in 2.1. On the other hand, theconcentration of functional monomer (3) is controlled (increased ordecreased depending on the desired density of dispersed phase); in thepresent case, 10% of AEM is added with respect to the monomer (1) NIPAM.

The characteristics of the polymer identified by the referencePNIPAM94), which was obtained using the operating method described in2.1, are given in the following table:

    ______________________________________                                        diameter.sup.(a), DLS 20° C. to 20° C.                                                  504 nm                                                  [sic]                                                                         diameter.sup.(b), DLS size at 40° C. 290 nm                            diameter.sup.(c), TEM 176 nm                                                  concentration of AEM.sup.(d) 22.4 μmol/g                                    of polymer                                                                   LCST.sup.(e) 32° C.                                                    CCC.sup.(f) at 20° C. 1.10 mol/1                                     ______________________________________                                         .sup.(a) diameter measured by dynamic light scattering at 20° C.       .sup.(b) diameter measured by dynamic light scattering at 40° C.       .sup.(c) diameter measured using electron microscopy                          .sup.(d) density of the dispersed phase expressed in mmol (primary            amine)/g of polymer.                                                          .sup.(e) lower critical solubility temperature (LCST) determined by           measuring the turbidity as a function of temperature                          .sup.(f) critical coagulation concentration (CCC) at 20° C.       

EXAMPLE 2 Synthesis and Characterization of the Ionic FerrofluidsIntended to be Incorporated into the Layer of the Second Polymer

The operating method was carried out, with the results mentioned in U.S.Pat. No. 4,329,241.

The physical properties of the ferrofluids produced according to thisdocument are given in the following summary table:

    ______________________________________                                        Properties Values         Methods                                             ______________________________________                                        diameters (nm)                                                                           15 ± 3      AFM (atomic force                                     and  microscopy)                                                              dispersity 7 ± 1 TEM (transmission                                           electron microscopy)                                                         9.5 ± 2   magnetization                                                     (measurement of the                                                           magnetization)                                                               11 ± 1  XR (X-rays)                                                       thickness of the 0.1 magnetization                                            non-magnetic                                                                  layer (nm)                                                                    specific magne- 422 kA/m magnetization                                        tization                                                                      charge density 1.5 conductimetry                                              (C/m.sup.2)                                                                   pH 7-8 pH measure-                                                              ment                                                                        conductivity 1 mS conductimetry                                             ______________________________________                                    

The method of synthesis, by the precipitation of iron oxides, makes itpossible, from the results of the various methods of characterization,to obtain an anionic ferrofluid which is stable between pH 6 and pH 8,has a size of the order of ten nm and is superparamagnetic. The analyseswere carried out for the various ferrofluids obtained using the sameoperating method: the results obtained are reproducible from oneferrofluid to another.

Since the ferrofluid particles are negatively charged, it is thereforepossible for these particles to be adsorbed on a polymer of oppositecharge (positively charged) via electrostatic interactions.

EXAMPLE 3 Adsorption of the Magnetic Filler on the Layer of SecondPolymer

The second polymer and the magnetic filler (Example 2) have charges ofopposite sign. This promotes strong adsorption, mainly via electrostaticinteractions, of the magnetic filler on the polymer. The magnetic filleris placed in excess with respect to the concentration of polymer(Example 1) and the adsorption is carried out under conditions such thatthe degree of coverage of the surface of the second polymer is greaterthan 30%. 6.4 ml of the sol of the polymer obtained in Example 1 (1.2;concentration=94.5 g/l) are progressively added, in a 200 ml flask, to29 ml of the ferrofluid obtained in Example 2 (concentration=23 g/l).After adsorption of the ferrite for 15 minutes, the excess ferrofluid isremoved by placing the flask on a magnet so as to separate the polymerparticles covered with ferrite. The supernatant liquid is removed,assayed and replaced by the same volume of boiled and degassed water.The flask containing the polymer covered with ferrite is again placed onthe magnet so as to check that no ferrite in solution remains (clearsupernatant liquid).

The table presented below gives the amount of ferrite adsorbed on thehairy latex particles of Example 1.1:

    ______________________________________                                                   Amount adsorbed                                                      Code in g/g of latex mass % adsorbed                                        ______________________________________                                        ENC10      6.63        45                                                       ENC11 6.67 46                                                                 ENC13 4.70 40                                                                 ENC12 5.30 37                                                                 ENC16 5.00 40                                                               ______________________________________                                    

EXAMPLE 4 Encapsulation of the Particles

The process of encapsulating the magnetic filler after the adsorptionstep consists in polymerizing, in aqueous medium, one or more monomerswith a copolymerizable crosslinking agent, in the presence of asaturated suspension of magnetic polymer (Example 3). The monomerschosen may be functional and thus, in this case, be useful for thegrafting or adsorption of biological molecules. This method makes itpossible to obtain a magnetic polymer whose interface may be easilymodified depending on the uses: a hydrophobic surface for the adsorptionof a protein or a functional hydrophilic surface for chemical grafting,for example.

The encapsulation process described in this example relies on the use ofan initiator and of a mixture of monomers and of a crosslinking agent.40 ml of ferrite-covered polymer (Example 3) are introduced into areactor thermostatted at 70° C. The monomers are then introduced into a4 ml volume of boiled and degassed water. The polymerization time isthree hours starting from the introduction of the initiator. Themonomers used are indicated in the following tables:

4.1)

    ______________________________________                                        Reactants              Quantities                                             ______________________________________                                        NIPAM                  0.15 g                                                   MBA 0.0075 g                                                                  Potassium persulfate (KPS) 0.0056 g                                         ______________________________________                                    

The magnetic particles obtained have the references: ENC10, ENC11 andENC13.

4.2)

    ______________________________________                                        Reactants              Quantities                                             ______________________________________                                        NIPAM                  0.15 g                                                   MBA 0.015 g                                                                   Potassium persulfate (KPS) 0.0056 g                                         ______________________________________                                    

The magnetic particles obtained have the references: ENC12 and ENC16.

These particles were characterized. The results are given in thefollowing tables:

a) Diameter of the magnetic particles obtained

    ______________________________________                                        Code      D.sup.(a) nm                                                                          Dn.sup.(b) nm                                                                              Dw.sup.(b) nm                                                                        DI.sup.(c)                              ______________________________________                                        ENC10     500     380          388    1.02                                      ENC11 810 385 388 1.006                                                       ENC13 1500 352 363 1.030                                                      ENC12 750 366 369 1.007                                                       ENC16 1000 ND ND ND                                                         ______________________________________                                         .sup.(a) diameter measured by dynamic light scattering                        .sup.(b) diameter measured by transmission electron microscopy                .sup.(c) polydispersity index                                                 ND not determined.                                                       

b) Mass percentage of ferrite encapsulated in the polymer and theseparation time

    ______________________________________                                               mass % of ferrite                                                                          mass % of ferrite                                                                          separation time                                Code magnetization complexometry min (*)                                    ______________________________________                                        ENC10  35           43           <20                                            ENC11 23 40 <20                                                               ENC13 38 36 <20                                                               ENC12 16 23 <30                                                               ENC16 40 45  <5                                                             ______________________________________                                         (*) The separation time is determined using a GENPROBE magnet, Magnetic       separation unit, catalog #1639, San Diego.                               

The colloids obtained are stable, monodisperse and have a separationtime, under the action of a magnetic field, of less than 30 min, andmore particularly less than 5 min (ENC16). The sizes obtained arereproducible from one synthesis to another and are between 0.1 and 10μm, and the size distribution within the same preparation is very narrow(DI<1.03). The amount of ferrite encapsulated is between 40 and 45%. Theencapsulation does not cause desorption of the magnetic particles sinceit has been checked that the difference between the adsorbed masspercentage and the mass percentage after encapsulation is very small.

What is claimed is:
 1. Monodisperse superparamagnetic particles having apredetermined size of between 0.1 and 10 μm, comprising:a core based ona first polymer, an internal layer, called the magnetic layer, coveringthe core, based on a second polymer, wherein in said internal layer amagnetic material is distributed, and an external layer, called theencapsulation layer, optionally functionalized, covering the magneticlayer, based on a third polymer and having an ability to interact withat least one biological molecule to fix the at least one biologicalmolecule to the particles, either directly or indirectly, wherein atleast the second polymer is heat-sensitive and has a predetermined lowercritical solubility temperature (LCST) of between 15 and 65° C.
 2. Theparticles according to claim 1, wherein the second polymer is obtainedby polymerization of (1) a water-soluble monomer of acrylamide or of anacrylamide derivative, (2) at least one crosslinking agent and (3) atleast one functional, cationic and water-soluble monomer different fromthe monomer (1).
 3. The particles according to claim 2, wherein thesecond polymer is poly(N-isopropylacrylamide) (PNIPAM) obtained bypolymerization of (1) N-isopropylacrylamide, (2)N,N-methylenebisacrylamide and (3) 2-aminoethyl-methacrylate chloride.4. The particles according to claim 2, wherein the first polymer isidentical to the second polymer.
 5. The particles according to claim 1,wherein the first polymer is different from the second polymer and is apolymer having a hydrophobic character.
 6. The particles according toclaim 1, wherein the third polymer is a hydrophilic polymer.
 7. Theparticles according to claim 6, wherein the third polymer isfunctionalized and bears a functional group selected from the groupconsisting of carboxylic, aldehyde, thiol and amine functional groups.8. The particles according to claim 1, said particles having anessentially spherical shape.
 9. A process for obtaining particles asdefined according to claim 1, comprising:(a) obtaining the first polymerby polymerization of the suitable monomer or monomers, (b) obtaining asol of the second polymer by polymerization in aqueous phase of (1) awater-soluble monomer of acrylamide or of an acrylamide derivative, (2)at least one crosslinking agent and (3) at least one functional,cationic and water-soluble monomer different from the monomer (1), (c)bringing the magnetic material into contact with the first and secondpolymers at a temperature below the lower critical solubilitytemperature (LCST) of the second polymer to adsorb the magneticmaterial, (d) bringing the reaction mixture obtained in (c) to atemperature above the LCST of the second polymer to obtain the magneticlayer, and (e) bringing the mixture obtained in (d) into contact, inaqueous phase, with the monomer or monomers suitable for obtaining thethird polymer by polymerization to encapsulate the mixture obtained in(d).
 10. The process according to claim 9, wherein steps (a) and (b) arecarried out simultaneously.
 11. The process according to claim 10,wherein the first polymer is identical to the second polymer.
 12. Theprocess according to claim 9, wherein the first polymer is differentfrom the second polymer and is a polymer having a hydrophobic character.13. The process according to claim 9, wherein, for step (b) andoptionally step (a), the monomer (1) is selected from the groupconsisting of N-alkylacrylamides and N,N-dialkylacrylamides.
 14. Theprocess according to claim 13, wherein the monomer (1) is selected fromthe group consisting of N-isopropylacrylamide, N-ethylmethacrylamide,N-n-propylacrylamide, N-n-propylmethacrylamide,N-isopropylmethacrylamide, N-cyclopropylacrylamide,N,N-diethylacrylamide, N-methyl-N-isopropylacrylamide, andN-methyl-N-n-propylacrylamide.
 15. The process according to claim 9,wherein, for step (b) and optionally step (a), the functional monomer ormonomers (3) are selected from the group consisting of acrylic andmethacrylic derivatives, 2-aminoethyl-methacrylate (AEM) chloride,N-vinylpyridine derivatives, trialkylammonium derivatives andisothiouronium chloride derivatives.
 16. The process according to claim9, wherein, for step (b) and optionally step (a), the crosslinking agent(2) is water-soluble and is N,N-methylenebisacrylamide (MBA) or ethyleneglycol dimethacrylate.
 17. The process according to claim 9, wherein thethird polymer is a hydrophilic polymer.
 18. The particles according toclaim 1, wherein the external layer has an ability to fix the at leastone biological molecule to the particles, directly or indirectly, byadsorption or covalent bonding.
 19. The particles according to claim 1,wherein said particles contain said magnetic material in an amount offrom 25 to 80% by weight.
 20. A process for isolating, in a liquidspecimen, at least one biological molecule, comprising:bringing saidspecimen into contact with particles according to claim 1, byincubation, wherein said particles interact with said at least onebiological molecule to fix the at least one biological molecule to theparticles directly or indirectly via a ligand thereon; applying amagnetic field to the mixture obtained; and separating the particlesfrom the specimen.
 21. The process according to claim 20, wherein saidat least one biological molecule is selected from the group consistingof proteins, antibodies, fragments of antibodies, antigens,polypeptides, enzymes, haptens, nucleic acids and fragments of nucleicacids.
 22. The process according to claim 20, in which the at least onebiological molecule is fixed to the particles, directly or indirectly,by adsorption or by covalent bonding.
 23. A reactive medium for theisolation of biological molecules, the reactive medium comprising adispersion, in aqueous medium, of particles as defined in claim 1.